The effects for inflammatory responses by CPP with different colloidal properties in hemodialysis patients

Calciprotein particles (CPPs) are colloids composed of solid-phase calcium-phosphate and serum protein fetuin-A. CPPs form a polydispersed system with different particle size and density. CPPs with specific physical properties can induce calcification and innate immune responses in cultured cells. In hemodialysis patients, blood CPP levels were reported to correlate with vascular calcification and inflammation. However, little is known about relation between these disorders and physical properties of CPPs. Here, we show that the association between physical properties of plasma CPPs and serum levels of inflammatory cytokines/chemokines in 78 hemodialysis out-patients by cross-sectional study. Patients with cardiovascular disease (CVD) had significantly higher high density CPP (H-CPP) levels than patients without CVD but not low density CPP (L-CPP). Seven cytokines/chemokines (EGF, eotaxin, IL-8, IP-10, MCP-1, MIP-1, MIP-1β and TNFα) were detectable in the serum samples from > 95% of the patients. In multivariate regression analysis, H-CPP was positively associated with eotaxin after adjusting for age, gender, smoking, serum phosphate and FGF23. L-CPP was negatively associated with IL-8 after adjusting for age, gender, serum albumin, phosphate and FGF23. High H-CPP levels were associated with pro-inflammatory response, whereas L-CPPs were associated with anti-inflammatory response. CPPs with different physical properties may impact differently on pathophysiology in HD patients.


Materials and methods
Patients and study design. Circulating levels of CPPs, cytokines and chemokines were measured in 78 clinically stable HD out-patients recruited from a clinic (Seiikai medical clinic Oyama, Tochigi, Japan) between October 2016 and November 2016. Exclusion criteria were age < 18 years, signs of overt clinical infection and unwillingness to participate. All patients were treated with the use of high-flux membranes 27 , and the median blood flow was 230 (10-90th percentile 200-300) ml/min, the median duration of each dialysis session was 4.5 (10-90th percentile 3.8-5.5) hours, and the calcium level of the dialysate of all patients was 3.0%. Ultrapure dialysis fluid was managed in according to the standards of Japanese Society for Dialysis Therapy. Written Informed consent was obtained from each patient. The Ethics Committee in Jichi Medical University approved study protocols. The studies were conducted in adherence with the Declaration of Helsinki.

Quantification of plasma calciprotein particles (CPPs). Circulating CPP levels have been quantified
by the "fetuin-A method": Serum/plasma samples are centrifuged at 16,000 g for 2 h to precipitate CPPs. The difference in the serum/plasma fetuin-A levels determined by ELISA between before and after the centrifugation is assumed to represent the CPP level. Thus, the fetuin-A method measures CPPs over a certain density that can be precipitated by the centrifugation at 16,000 g for 2 h. On the other hand, we recently developed a novel CPP assay termed "gel-filtration method" 18 and found that decent amount of secondary CPPs were present in the supernatant of serum/plasma samples centrifuged at 16,000 g for 2 h. These CPPs were designated as L-CPPs. The difference in the fluorescence intensity before and after the centrifugation correlated with the CPP level determined by the fetuin-A method and designated as H-CPPs.
In the present study, CPPs in blood samples were quantified by the gel-filtration method. Preparation of plasma samples was strictly standardized to avoid potential variation of the amorphous-to-crystalline phase transition of CaPi in CPPs in vitro. Blood was drawn immediately before dialysis at two-days of interval using heparinized blood collecting tubes. The blood samples were centrifuged at 3000 rpm for 10 min to separate plasma within 60 min after sampling. The plasma samples were aliquoted in microcentrifuge tubes, snap-frozen in liquid nitrogen, and stored at − 80 °C. The frozen plasma samples were thawed 24 h before starting the CPP assay and incubated at 25 °C to convert amorphous CaPi in CPPs to crystalline CaPi. After incubation for 22 h, each sample was divided into two tubes. One of the tubes was centrifuged at 16,000 g for 2 h at 25 °C. The supernatant was used for measurement of theL-CPPlevel. The other tube was left untreated at 25 °C for 2 h and then used for measurement of the total CPP (T-CPP) level.
A fluorescent probe that binds to CaPi crystals (OsteoSense 680EX; PerkinElmer Inc., Waltham, MA) was added to plasma samples. After incubation at 25 °C for 60 min, the sample was applied to a gel-filtration spin column to remove unbound OsteoSense. The amount of CPPs in the sample was expressed as the fluorescence intensity of the flow-through fraction quantified using an infrared fluorescence scanner (Odyssey CLx; LICOR Biosciences, Lincoln, NE). The H-CPP level was calculated by subtracting the L-CPP level from the T-CPP level. In case that calculated H-CPP was less than zero, the H-CPP level was designated as zero. www.nature.com/scientificreports/ Blood sampling and laboratory analysis. Serum levels of albumin, triglycerides, total cholesterol, high-density lipoprotein (HDL)-cholesterol, calcium, phosphate, C-reactive protein, plasma intact-parathyroid hormone (PTH) and hemoglobin were analyzed using certified methods at the clinic. Low-density lipoprotein (LDL)-cholesterol was calculated using the Friedewald formula: [(total cholesterol)-(HDL-cholesterol)-(triglycerides/5)] 28 . Serum intact fibroblast growth factor 23 (FGF23) levels were measured using an enzyme-linked immunosorbent assay kit (Kainos Laboratories, Inc., Tokyo, Japan).

Measurements
Clinical assessments. Presence of CVD was defined as history or signs of ischemic heart disease, peripheral vascular disease, and/or cerebrovascular disease. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters.
Statistical analyses. Data are expressed as median (10th to 90th percentile) or percentage. Statistical significance was set at the level of p < 0.05. Comparisons between two groups were performed by non-parametric Wilcoxon test for continuous variables and Chi-square test for nominal variables. Non-parametric Spearman rank correlation analysis was used to determine associations between variables. Multiple linear regression analyses were performed for continuous variables of H-CPPs and L-CPPs. The results were shown as standardized β regression coefficients. Parameters showing significant association in bivariate analyses were used for adjustment in multiple linear regression analyses. Statistical analyses were performed using statistical software JMP 14 (SAS institute Inc., Cary, NC, USA) and Stata 16.0 (Stata Corporation, College Station, TX, USA).

Results
Clinical and laboratory characteristics. Clinical and biochemical characteristics of 78 HD out-patients are presented in Table 1. Their ages ranged from 26 to 78 years. The median age of the patients was 62 years, 67% were males. 41% had diabetes mellitus (DM). The etiologies of renal disease were chronic glomerulonephritis (n = 22; 28%), hypertension and renovascular disease (n = 6; 8%), polycystic kidney disease (n = 9; 11%), diabetic nephropathy (n = 28; 36%) and others or unknown causes (n = 13; 17%). The median dialysis vintage was 59 months. Clinical signs or symptoms of CVD were present in 18% of the patients (cerebrovascular disease: 5 patients, ischemic heart disease: 5 patients, and peripheral artery disease: 6 patients). The median plasma T-CPP, L-CPP and H-CPP levels were 279,006, 149,838 and 124,418 AU, respectively.

Association of CPPs with the prevalence of CVD. Plasma H-CPP levels were positively correlated
with T-CPP levels, but not with L-CPP levels (Fig. 1A, B), indicating that the increase in T-CPP levels could be explained primarily by the increase in H-CPP levels, but not L-CPP levels. Accordingly, the ratio of L-CPP to T-CPP was significantly decreased as the T-CPP or H-CPP levels were increased (Fig. 1C, D). Patients with CVD had significantly higher H-CPP levels and tended to have higher T-CPP levels than patients without CVD. However, L-CPP levels were not different between patients with or without CVD (Fig. 2).

Multivariate analysis.
Multivariate regression models were used to determine independent significant predictors of H-CPP and L-CPP (

Discussion
The present study demonstrated that CPPs with different colloidal properties were correlated with different clinical parameters and inflammatory responses. First, plasma H-CPP levels associated positively with serum levels of eotaxin. Second, plasma L-CPP levels associated negatively with serum IL-8 levels after adjusting with possible co-variates. Gatate et al. demonstrated that plasma T-CPP levels were associated with CVD events in hemodialysis patients under a prospective design 14 . Because T-CPP levels were positively correlated with plasma H-CPP levels (Fig. 1), we expected to observe that patients with CVD should have higher H-CPP and T-CPP levels than patients without CVD. As expected, we observed that the patients with CVD had higher H-CPP levels than the patients without CVD (Fig. 2). However, the difference in the T-CPP levels did not reach statistical significance (p = 0.06). This may be due to the small number of patients enrolled in the present study and the different (prospective vs cross-sectional) study design. Further studies are required to determine the association of CPPs with different physical property with clinical outcomes. www.nature.com/scientificreports/ The eosinophil chemokine eotaxin (also known as CCL11) is highly expressed in human atherosclerotic plaques 29 . An increase in serum eotaxin levels were reportedly associated with coronary artery disease, suggesting that eotaxin may contribute to vascular inflammation [29][30][31] . In addition, we found negative correlation between L-CPPs and serum levels of IL-8, which is known to participate in pathogenesis of CVD 32 . Prospective clinical studies demonstrated that an increase in plasma IL-8 concentration was associated with coronary artery calcifications, identifying IL-8 as a powerful prognostic predictor of all-cause and cardiovascular mortality in CKD patients 33,34 . In the present study, serum IL-8 levels were correlated positively with serum β2-microglobulin levels and negatively with serum albumin levels (Supplementary Table 2). IL-8 may be one of the inflammatory mediators linking between uremic toxin, malnutrition, and vascular calcification in CKD. Recently, Perna et al. reported that increased plasma levels of eotaxin and IL-8 were associated with low GFR and vascular calcification 35 . These findings indicate that H-CPPs and L-CPPs may exert opposite effects on inflammation. Namely, it is intriguing to speculate that H-CPPs and L-CPPs may behave like pro-inflammatory and antiinflammatory factors, respectively.
Eotaxin expression is upregulated in tissues with allergic inflammation and associated with eosinophil infiltration and disease severity 36,37 . Eotaxin mRNA levels were reported to be increased not only in the skin of atopic dermatitis patients but also in biopsies from itchy skin lesions when eosinophils are present 38 . The immune system dysregulation and inflammation are one of the possible mechanisms for generation of pruritis 39 . Chronic kidney disease-associated pruritus (CKD-aP) is a common symptom and impairs Health-Related Quality of Life (HRQoL) and clinical outcomes in patients undergoing dialysis [40][41][42][43] . Increased eosinophils have also been observed in non-dialysis patients with CKD-aP 44 . Keithi-Reddy et al. reported a case of uremic pruritus who had eosinophilia and eosinophil infiltration in the skin biopsy 45 . The pathophysiology of CKD-aP is complex and incompletely understood. A hypothesis on the mechanism of CKD-aP implicated toxins which include calcium, phosphate and magnesium in the skin and subcutaneous tissue as potential pruritogens. This hypothesis was based on several observations,e.g., the association of CKD-aP with higher calcium and phosphate levels,and the improvements in pruritus after treatment of high calcium and phosphate levels. However, subsequent studies have not confirmed these associations 46 . Thus, no information on the possible involvement of H-CPPs and eotaxin in CKD-aP is available so far. Further studies are required to clarify the roles that CPPs, eotaxin and eosinophils may play in pathogenesis of CKD-aP. www.nature.com/scientificreports/ Limitations of the present study include the relatively low number of participants and its observational cross-sectional design that does not permit conclusions concerning causality. Second, we did not investigate several other potential determinants of eotaxin level, such as immunoglobulin E (IgE), eosinophil count, and symptoms of pruritus, which are common in HD patients. Third, we explored only cytokines/chemokines which were detected by using Milliplex® map 29 premix kit. Using other measuring method could have identified other cytokines/chemokines associated with CPPs. However, this method has the advantage of assessing a large number of immune modulators simultaneously, which enabled us to observe different aspects of immune response.  Table 2. bivariate correlations expressed as rho correlations of EGF, Eotaxin, IL-8, IP-10, MCP-1, MIP-1β and TNFα with CPPs at baseline in 78 HD patients. Signigficant values are in bold. T-CPP total calciprotein particle; L-CPP low density calciprotein particle; H-CPP High density calciprotein particle; EGF epidermal growth factor; IL-interleukin-; IP-10 Interferon gamma-induced protein 10 (C-X-C motif chemokine ligand 10); MCP-1 Monocyte chemoattractant protein − 1; MIP-1α macrophage inflammatory protein 1α; MIP-1β Macrophage inflammatory protein-1β; TNFα tumor necrosis factor α.